Erythropoietin (Epo) and thrombopoietin (Tpo) are the primary cytokines regulating red blood cell production, and megakaryocyte and platelet development, respectively. The adaptor protein Lnk negatively regulates signaling transduction of the Epo receptor (EpoR) and Tpo receptor (mpl), thereby downregulating Tpo-mediated megakaryocytopoiesis and Epo-mediated erythropoiesis. Lnk inhibits the activity of cytokine receptor integral partner, the JAK2 tyrosine kinase. Furthermore, the Lnk SH2 domain is essential for its inhibitory functions, and the functional interaction between Lnk and the receptor/JAK2 complex is phosphorylation-dependent. The purpose of this grant is to unravel the mechanism that encodes how Lnk controls the quantitative response to cytokines, and whose dysregulation predisposes to leukemia. To accomplish this, biochemical, cellular, and mouse genetic approaches will be used with the focus on the Lnk SH2 domain and tyrosine residues. Recombinant peptide library screens will establish Lnk SH2 domain binding motifs and mass spectrometry (MS) will identify Lnk SH2 domain interacting proteins in vivo. Novel primary hematopoietic cell culture systems will be developed to investigate the interactions among Lnk, cytokine receptors, and JAK2. Moreover, the Lnk tyrosines that are phosphorylated in vivo and important for its function will be dissected. Importantly, the mechanisms of Lnk regulatory function will be tested within physiological contexts using gene-deficient primary hematopoietic cells and generating gene-ablated mice. Unchecked and sustained cytokine receptor signaling leads to oncogenic transformation, therefore understanding the cellular mechanisms by which Lnk controls the amplitude and duration of receptor signaling will shed significant light on cytokine receptor regulation in general. Through this proposed research, the applicant will receive training in structural and proteomics biology and will gain the necessary skills and knowledge to become an independent investigator.